Methods for resolving lipoproteins with mass spectrometry

ABSTRACT

The present disclosure relates to methods of identifying components present in intact lipoprotein particles. Methods provided include single particle mass spectrometry, such as charge detection mass spectrometry (CDMS). Distinct subtypes and subpopulations that exist within lipoprotein density classes are determined based on simultaneously measured m/z and charge of ionized lipoprotein particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/647,955, filed Mar. 17, 2020, which is a national stage entry ofInternational Patent Application No. PCT/US2018/051944, filed Sep. 20,2018, which claims the benefit of and priority to U.S. ProvisionalPatent Application No. 62/561,184 filed Sep. 20, 2017, the disclosuresof which are expressly incorporated herein by reference in theirentireties.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under CHE1531823 awardedby National Science Foundation. The government has certain rights in theinvention.

BACKGROUND

Lipoproteins are involved in cholesterol transport in human plasma, havea diverse range of metabolic function, and serve as biomarkers forcoronary artery disease. Lipoproteins are separated into classes basedon the density of protein relative to the lipid composition, includinghigh-density (HDL), low-density (LDL), and very low-density (VLDL). HDLis commonly referred to as the “good cholesterol,” while high amounts ofLDL, or “bad cholesterol” have been implicated in coronary arterydisease. There is an inverse correlation of HDL particle concentrationand cardiovascular disease. (Castelli, W. P., et al. Circulation. 1977(55) 767-772, incorporated by reference herein).

Each density class can be split into several subtypes which differ insize, composition, and metabolic properties. Furthermore, each subtypecontains particles that differ in number of structural proteins, givingrise to many possible protein/lipid combinations that encompass a rangeof unique structures. The major density classes are linked through aseries of lipid and protein exchange and conversion during metabolism,causing great variation in particle mass for a single population.Information about the many subtypes and diverse subpopulations of HDL iscurrently incomplete (Karathanasis, S. K., et al. Clin. Chem. 2017 (63)196-210, incorporated by reference herein).

Characterizing macromolecular complexes is challenging because theresolution of conventional mass analyzers is not sufficient to assigncharge states in the mass-to-charge (m/z) spectrum for heterogeneous andhighly charged complexes, such as lipoproteins. Peaks in the m/zspectrum broaden and shift due to mass heterogeneity, either intrinsicor due to complex formation. Poorly resolved peaks in the m/z spectrumprevent charge state assignment and mass determination. Evensub-megadalton (MDa) sized complexes can contain hundreds of charges,making it a challenge to accurately determine mass.

While the development of new therapies for improved cardiovascularhealth would benefit from the development of techniques to measure HDLand LDL subtypes directly, particle diversity and heterogeneity impedecharacterization by analytical techniques known in the art. Thus, thereis a need for new methods for analyzing lipoproteins.

SUMMARY

The present disclosure provides methods for resolving lipoproteins usingsingle particle mass spectrometry, such as charge detection massspectrometry (CDMS). Distinct subpopulations that exist withinlipoprotein classes are revealed by correlating m/z and mass (or byother correlations involving mass, charge and m/z measurements).

According to one aspect of the present disclosure, a method ofidentifying components present in a lipoprotein includes subjecting thelipoprotein to mass spectrometry to produce ions, measuring a charge ofeach ion, measuring a mass-to-charge ratio of the ion, and identifyingthe components based on the mass of the lipoprotein.

In some embodiments, the charge of the ion and the mass-to-charge ratioof the ion may be measured simultaneously. In some embodiments, thecharge of the ion and the mass-to-charge ratio of the ion may bemeasured using charge detection mass spectrometry. In some embodiments,the charge detection mass spectrometry may include focusing the ion intoa linear ion trap, the linear ion trap comprising a charge detectioncylinder. In some embodiments, the charge detection mass spectrometrymay include focusing the ion into a linear ion trap and the linear iontrap incorporates a charge detection cylinder. In some embodiments, thelinear ion trap may be a cone trap.

In some embodiments, the mass-to-charge ratio of the ion may be measuredbased on a time period that the ion takes to traverse the chargedetection cylinder or the fundamental frequency at which the ionoscillates in the charge detection cylinder.

In some embodiments, the charge of the ion may be measured based on theamplitude of a signal due to the ion oscillating in the charge detectioncylinder. In some embodiments, the ion may be trapped in the linear iontrap for a trapping period to determine the mass to charge ratio of theion with sufficient accuracy. For example, the ion may be trapped in thelinear ion trap for a trapping period of about 50 ms to about 150 MS.

In some embodiments, the lipoprotein may include one or more ofhigh-density lipoprotein particles (HDL), low-density lipoproteinparticles (LDL), and very low density lipoprotein particles (VLDL). Insome embodiments, the method may further include estimating the diameterof a particle of the lipoprotein based on the mass of the ion and thedensity of a known lipoprotein subtype particle. In some embodiments,the lipoprotein may include a plasma lipoprotein, and the ion may be inthe presence of one or more other lipoprotein particles.

In some embodiments, the mass of the ion may be within about 1 MDa ofone or more other lipoprotein particles having a different subtypecompared to the ion. In other embodiments, the mass of the ion may bewithin about 100 kDa of one or more other lipoprotein particles having adifferent subtype compared to the ion. Yet, in some embodiments, themass of the ion may be within about 20 kDa of one or more otherlipoprotein particles having a different subtype compared to the ion. Insome embodiments, the ion may be an intact lipoprotein particle. In someembodiments, subjecting the lipoprotein to mass spectrometry to produceions may include subjecting the lipoprotein without isolating alipoprotein particle prior to subjecting the lipoprotein to massspectrometry to produce an ion.

In some embodiments, the mass of the ion may be from about 100 kDa toabout 80 MDa. In some embodiments, the mass of the ion may be from about10 MDa to about 80 MDa. In some embodiments, the mass of the ion may befrom about 100 kDa to about 3 MDa. In some embodiments, the method mayinclude subjecting a single sample to a mass spectrometer.

According to another aspect of the present disclosure, a method forevaluating a patient's risk of cardiovascular disease includesidentifying components present in a lipoprotein according to any of thepreceding clauses. In some embodiments, identifying components presentin a lipoprotein may include identifying components present in alipoprotein in a blood sample.

According to another aspect of the present disclosure, a method ofidentifying components present in a lipoprotein includes correlating m/zand charge, or quantities derived therefrom, to determine quantities ofHDL, LDL, and VLDL, wherein the lipoprotein is part of a samplecomprising serum.

According to another aspect of the present disclosure, a method ofidentifying components present in a lipoprotein includes correlating m/zand charge, or quantities derived therefrom, to determine subtypes ofHDL, LDL, and VLDL, wherein the lipoprotein is part of a samplecomprising whole blood.

According to another aspect of the present disclosure, a method ofidentifying components of a lipoprotein present in a sample includessubjecting the sample to mass spectrometry to produce ions, measuring acharge of each ion, measuring a mass-to-charge ratio of the ion, andidentifying the components based on the mass of the sample.

In some embodiments, the sample may include at least one of whole blood,plasma, or serum. In some embodiments, the charge of the ion and themass-to-charge ratio of the ion may be measured simultaneously. In someembodiments, the charge of the ion and the mass-to-charge ratio of theion may be measured using charge detection mass spectrometry. In someembodiments, the charge detection mass spectrometry may include focusingthe ion into a linear ion trap, the linear ion trap comprising a chargedetection cylinder. In some embodiments, the charge detection massspectrometry may include focusing the ion into a linear ion trap and thelinear ion trap incorporates a charge detection cylinder. In someembodiments, the linear ion trap may be a cone trap. In someembodiments, the mass-to-charge ratio of the ion may be measured basedon a time period that the ion takes to traverse the charge detectioncylinder or the fundamental frequency at which the ion oscillates in thecharge detection cylinder.

In some embodiments, the charge of the ion may be measured based on theamplitude of a signal due to the ion oscillating in the charge detectioncylinder. In some embodiments, the ion may be trapped in the linear iontrap for a trapping period to determine the mass to charge ratio of theion with sufficient accuracy. In some embodiments, the ion may betrapped in the linear ion trap for a trapping period of about 50 ms toabout 150 ms. In some embodiments, the lipoprotein may include one ormore of high-density lipoprotein particles (HDL), low-densitylipoprotein particles (LDL), and very low density lipoprotein particles(VLDL).

According to another aspect of the present disclosure, a method ofidentifying components present in a lipoprotein including subjecting thesample to mass spectrometry to produce ions, separating the ions basedon a mass-to-charge ratio, selecting a subset of the ions based on themass-to-charge ratio, dissociating the selected ions into fragments, andanalyzing the fragments to identify the components present in thelipoprotein.

In some embodiments, selecting the subset of the ions based on themass-to-charge ratio may include selecting a subset of the ions thatcorrespond to a subpopulation of the lipoprotein based on themass-to-charge ratio. For example, the mass-to-charge ratio maycorrespond to a subpopulation of the lipoprotein.

In some embodiments, selecting the subset of the ions based on themass-to-charge ratio may include selecting a subset of the ions using amass spectrometer. For example, the mass spectrometer may be atime-of-flight mass spectrometer, a quadrupole mass spectrometer, alinear and non-linear ion trap mass spectrometer, a Fourier transformmass spectrometer, a magnetic sector mass spectrometer, an Orbitrap massspectrometer, or a double focusing mass spectrometer.

In some embodiments, dissociating the selected ions into the fragmentsmay include dissociating the selected ions using a dissociation method.For example, the dissociation method may be collision induceddissociation, collisionally activated dissociation, surface induceddissociation, photo-induced dissociation, electron-impact induceddissociation, electron transfer dissociation, and/or electron capturedissociation. The photo-induced dissociation may further includeinfrared multiple photon dissociation and/or UV photodissociaton.

In some embodiments, analyzing the fragments to identify the componentspresent in the lipoprotein may include analyzing the fragments usingmass spectrometry and/or charge detection mass spectrometry. In someembodiments, the lipoprotein comprises one or more of high-densitylipoprotein particles (HDL), low-density lipoprotein particles (LDL),and very low density lipoprotein particles (VLDL).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a mass spectrum for HDL overlaid with a scatter plot of m/zversus mass where each point represents a single ion;

FIG. 1B is a mass spectrum for HDL-2 overlaid with a scatter plot of m/zversus mass where each point represents a single ion;

FIG. 1C is a mass spectrum for HDL-3 overlaid with a scatter plot of m/zversus mass where each point represents a single ion;

FIG. 2 is a mass spectrum for LDL overlaid with a scatter plot of m/zversus mass where each point represents a single ion;

FIG. 3 is a schematic diagram of a charge detection mass spectrometry(CDMS) apparatus used in connection with the present methods;

FIG. 4A is a mass spectrum measured for HDL overlaid with a scatter plotof m/z versus mass where each point represents a single ion. HDLsubclasses are resolved in the scatterplot;

FIG. 4B illustrates a mass distribution for HDL identical to that shownin FIG. 4A along with mass distributions for the HDL subpopulationsidentified in the scatterplot of m/z versus mass in FIG. 4A;

FIG. 4C illustrates mass distributions for the HDL subpopulationsoverlaid with scales giving approximate masses for HDL particles withn_(I) copies of Apolipoprotein A-I and n_(II) copies of ApolipoproteinA-II, wherein the large digits represent the number of Apo A-I and thesmall digits represent the number of Apo A-II; and

FIG. 5 illustrates a mass spectrum measured for LDL overlaid with ascatter plot of m/z versus mass where each point represents a singleion. Subpopulations of LDL are resolved in the scatterplot. Note thatthese subpopulations have overlapping mass distributions.

DETAILED DESCRIPTION

The present invention will now be described with reference to theaccompanying drawings, in which representative embodiments of theinvention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference herein intheir entirety.

The present disclosure provides methods for resolving lipoproteins bymeasuring the mass-to-charge ratio (m/z) and charge (z) of lipoproteinions passing through a mass spectrometer. In some embodiments, thesemethods are used to characterize high-density (HDL) and low-density(LDL) lipoprotein particles, even when the particles are highlyheterogeneous.

It is to be understood that lipoproteins may comprise micellar molecularstructures having several structural proteins and lipid molecules.Lipoproteins may comprise tens of hundreds of lipid molecules.

HDL is comprised of approximately 50% protein by mass, of which, twomajor structural proteins apolipoprotein AI (Apo-AI) and apolipoproteinAII (Apo-AII), contribute to about 70% and about 20% of the totalprotein content, respectively. The additional 10% can be attributed toenzymes and lipid transfer proteins, as well as >5% corresponding tominor proteins. Lipid transfer proteins are mainly responsible for theexchange of lipids between lipoprotein particles, and thus, contributeto the unfixed masses of HDL particles. The primary contributor toheterogeneity of HDL is the conformational flexibility of the mainstructural protein Apo-AI. It dictates particle structure throughscavenging phospholipids and thus driving the formation of small,medium, and large particles. As particle size increases, the number ofApo-AI proteins also increases; some HDL subtypes carry more than 4Apo-AI molecules per particle. In addition, Apo-AI has the capacity tobind and interact with additional proteins where specificprotein-protein complexes have been observed to form with HDL particles.Over 50 accessory proteins and more than 200 individual lipid specieshave been identified in human HDL structure and transport. Withouthaving a fixed stoichiometry, combined with the dynamic nature ofexchangeable proteins, it may be difficult to parse out a fixed mass foreach subtype. In some embodiments, a range of masses and sizes may beused to broadly characterize the observed subpopulations within HDL andits subtypes.

The high density subtypes HDL-2 and HDL-3 can be separated further intofive distinct subpopulations HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c. Eachsubpopulation differs in size and protein composition, with the maindifferences between HDL-2 and HDL-3 subtypes corresponding to theirlipid rich and lipid-poor construction, respectively.

Low density lipoproteins are greater in size than high densityparticles; the diameter of LDL is at least twice as large as HDL andincorporates hundreds more lipid molecules. The low density lipoproteinclasses suffer from inherent heterogeneity due to the high ratio oflipids and cholesterol relative to protein. Larger VLDL is converted toLDL in the bloodstream through a series of delipidation steps.Structural protein apolipoprotein-B100 (apoB-100) is non-exchangeableand does not vary in terms of number of molecules per particle. Instead,there are several other ancillary proteins that comprise LDL that aredynamic and can be exchanged during cholesterol transport.

The methods of the present disclosure can be employed to establish themass and stoichiometry of lipoprotein particles. In some embodiments,the methods described herein can be employed to distinguish betweenvarious lipoprotein particle subtypes. Subpopulations and subtypes thatexist within lipoprotein classes may be identified by correlating m/z,charge, and mass to reveal distinct subpopulations that exist withinlipoprotein classes and subtypes that would be difficult or impossibleto observe by ensemble techniques alone. In some embodiments, thepresent methods are used to observe low abundant subpopulations thatexist within subtypes of HDL. The methods of this disclosure can also beused to resolve differences in lipoprotein structure, morphology,orientation, size, chemical status, and the like. The lipoproteinparticles used in connection with the present disclosure may compriseintact lipoprotein particles. Additionally, the lipoprotein particlesmay be analyzed without the use of isolation or without the use ofextensive isolation.

It is to be understood that the masses of the lipoprotein particles usedin connection with the present methods may range from about 100kilodaltons (kDa) to about 10 megadaltons (MDa), about 100 kilodaltons(kDa) to about 5 megadaltons (MDa), about 100 kilodaltons (kDa) to about3 megadaltons (MDa), or about 100 kilodaltons (kDa) to about 2megadaltons (MDa), or about 10 megadalton (MDa) to about 80 megadaltons(MDa).

In some embodiments, many lipoprotein classes are present in a sampleused in connection with the methods described herein. In someembodiments, the methods comprise subjecting a single sample to a massspectrometer. In some embodiments, samples containing isolated subgroupsare measured as standards to aid in characterizing particle propertiessuch as stoichiometry.

Analytical methods described herein include single particle massspectrometry, such as charge detection mass spectrometry (CDMS), whichis a single particle technique that measures the mass-to-charge ratio(m/z) and charge (z) for each individual ion. Single particle massspectrometry may allow for differentiating heterogeneous species in themass spectrum by measuring, such as simultaneously measuring, m/z and zand directly deducing mass therefrom. Multiplying m/z and z gives themass of a particle directly. In addition, the measured mass and knowndensity information can be used to estimate the particle diameter. Thisinformation may be used for characterizing lipoprotein subtypes andsubgroups that vary slightly in size and protein composition.

For example, single particle mass spectrometry methods described hereinare capable of resolving HDL subtypes and subgroups within heterogeneousHDL samples by correlating the mass and m/z measurements (or by othercorrelations involving mass, charge and m/z measurements). The singleparticle measurement afforded by single particle mass spectrometryallows for low abundance populations to be observed. The ability tomeasure single particles one at a time allows for the measurement of lowabundance species within the mixture of particles. Single particle massspectrometry is also capable of providing the accurate mass of intactlipoprotein particles.

Nonlimiting examples of single particle mass spectrometry approachesthat can be employed in the methods of this disclosure include time offlight mass spectrometry with a cryogenic detector, charge detectionmass spectrometry (CDMS), quadrupole ion trap mass spectrometry withoptical detection and charge detection, Fourier transform ion cyclotronresonance, Orbitrap mass spectrometry and micromechanical/nanomechanicaloscillators. A detailed description of various single molecule massspectrometry approaches included in this disclosure can be found inKeifer & Jarrold (“Single molecule mass spectrometry” Mass SpectrometryReviews; DOI 10.1002/mas.21495 (2016) Wiley Periodicals, Inc.; theentire contents of which are incorporated by reference herein). In someembodiments, information on lipoprotein subtypes is obtained bycorrelating m/z and mass (or by other correlations involving mass,charge and m/z measurements). This information may be obtained from them/z spectrum alone if the results are calibrated by single particle massspectrometry, such as CDMS. In such embodiments, a conventional massspectrometer may be used to obtain information on subtypes oncestandards are calibrated, such as by CDMS.

In some embodiments, the methods described herein are used alongsideadditional techniques used to detect, quantify, and/or characterizelipoprotein composition. Additional techniques may comprise one or moreof liquid chromatography, nuclear magnetic resonance, size exclusionchromatography, electrofiltration or two-dimensional electrophoreticmobility, and related proteomics approaches.

Charge detection mass spectrometry (CDMS) is a single particletechnique, where the m/z and z of individual ions are measuredconcurrently, thereby allowing direct determination of the mass of eachion. Examples of CDMS are described in Keifer et al. (Anal. Chem., 2015,87 (20), pp 10330-10337) and Pierson et al. (J. Am. Soc. Mass Spectrom.(2015) 26:1213-1220), which are incorporated by reference herein. Themethods described herein include using CDMS to analyze heterogeneousmixtures that are intractable by conventional MS methods.

Exemplary CDMS systems are described in Contino, N. C., et al., Int. J.Mass Spectrom. 2013, 345-347, 153-159; Contino, N. C.; et al., J. Am.Soc. Mass Spectrom. 2013, 24, 101-108; Pierson, E. E.; et al., Int. J.Mass Spectrom. 2013, 337, 50-56; Pierson, E. E.; et al., J. Am. Soc.Mass Spectrom. 2015, 26, 1213-1220; Keifer, D. Z.; et al., Anal. Chem.2015, 87, 10330-10337, incorporated by reference herein. In someembodiments, CDMS comprises passing ions through a conductive cylinder.The charge induced by the ion when it is in the cylinder is detected bya charge sensitive preamplifier. The time the ion takes to traverse thelength of the conductive tube is related to the m/z, while the amplitudeof the induced charge imparted onto the cylinder can be used todetermine z. In some embodiments, the detection cylinder is placedinside an ion trap so that an ion oscillates back and forth through thedetection cylinder many times, improving the signal to noise ratio.

Referring to FIG. 3 , one embodiment of a CDMS apparatus for carryingout the methods of the present disclosure is shown. The lipoproteinstandards are ionized via a commercial nanoelectrospray ionizationsource before entering the charge detection mass spectrometer. Ionsenter the instrument through a heated capillary and pass through an RFhexapole, an RF quadrupole, and an ion lens. Ions are energy selectedwithin a dual hemispherical deflection energy analyzer and then focusedinto a linear ion trap, such as a cone trap, that contains the chargedetection cylinder. To initiate a trapping event, a potential is placedon the rear end cap so that ions are reflected back through the trap.After a short delay, a potential is placed on the front end cap to closethe trap and cause the ion to oscillate back and forth through thecharge detection cylinder. The ion oscillating back and forth throughthe detection cylinder induces a periodic signal which is amplified,digitized, and then analyzed by a Fortran program using fast Fouriertransforms. The m/z is derived from the fundamental frequency and thecharge (z) is derived from the magnitude of the fundamental and firstharmonic.

In some embodiments, a trapping period in the linear ion trap may begreater than about 10 ms, about 25 ms, about 50 ms, about 75 ms, about100 ms, about 150 ms, about 200 ms, about 300 ms, about 400 ms, about 3s, or about 30 s. Additionally, the trapping time may be from about 10ms to about 1000 ms, about 25 ms to about 1000 ms, about 50 ms to about1000 ms, about 75 ms to about 1000 ms, about 100 ms to about 1000 ms,about 150 ms to about 1000 ms, about 200 ms to about 1000 ms, or about300 ms to about 1000 ms.

In some embodiments of the present disclosure, the single particle massspectrometry can be carried out or performed by time of flight massspectrometry, charge detection mass spectrometry, quadrupole ion trapmass spectrometry, Fourier transform ion cyclotron resonance and/orOrbitrap mass spectrometry. In some embodiments the single particle massspectrometry can be carried out or performed withmicromechanical/nanomechanical oscillator. These approaches for carryingout single particle mass spectrometry can be employed individually or inany combination.

In some embodiments, single particle mass spectrometry can be carriedout or performed on a commercial mass spectrometer retro-fitted forsingle particle measurements. As one nonlimiting example, a singleparticle detector can be retrofitted to an existing instrument (e.g., acommercial instrument) that would allow single particle massmeasurements to be performed. In one nonlimiting example, the commercialinstrument could be a quadrupole time of flight (QTOF) massspectrometer.

Sample preparation for carrying out the methods of this disclosure maybe carried out according to protocols described herein as well asprotocols known in the art for conventional mass spectrometry and singleparticle mass spectrometry methods. Such methods can involvetransferring a sample to a solution containing a volatile salt.

In some embodiments, volatile, structure preserving buffers areutilized. In some embodiments, the salt can be ammonium acetate,although other salts may be used in some embodiments. Ammonium acetatemay be present in a concentration of about 1 mM to about 15 mM, about 5mM to about 15 mM, about 10 mM to about 15 mM, about 1 mM to about 12.5mM, about 5 mM to about 12.5 mM, about 10 mM to about 12.5 mM, about 5mM, about 10 mM, or about 12.5 mM. In some embodiments, the buffer has apH of about 7 to about 8 or about 7.5.

In some embodiments, the methods of the present disclosure compriseanalyzing lipoproteins obtained from a patient to evaluate the patient'srisk for cardiovascular disease. The methods may comprise taking totalmeasures of HDL and LDL to evaluate a patient's risk for cardiovasculardisease. The HDL may comprise several subtypes with several subgroupsthat populate each subtype. Similarly, LDL may comprise of severalsubtypes. In some embodiments, the methods described herein comprisedirectly measuring subtypes and/or subgroups within a lipoproteinmixture. Such methods may be utilized to improve risk assessment throughcharacterization and/or identification of particular subgroups.

In some embodiments, tandem mass spectrometry or mass spectrometry/massspectrometry (MS/MS) techniques may be used to analyze lipoproteinsubpopulations. For example, ions with a narrow band of m/z values maybe selected using a mass spectrometer, and the ions may be broken intofragments using a variety of methods, such as, collision induceddissociation, collisionally activated dissociation, surface induceddissociation, photo-induced dissociation (including infrared multiplephoton dissociation and UV photodissociaton), electron-impact induceddissociation, electron transfer dissociation, and electron capturedissociation. The mass spectrometer may be a time-of-flight massspectrometer, a quadrupole mass spectrometer, a linear and non-linearion trap mass spectrometer, a Fourier transform mass spectrometer, amagnetic sector mass spectrometer, a Orbitrap mass spectrometer, adouble focusing mass spectrometer, or any other type of massspectrometer used for conventional mass spectrometry (MS). As discussedfurther below, CDMS measurements have shown that the lipoproteinpopulations and subpopulations have a narrow range of m/z values. Thisallows a specific subpopulation of lipoprotein to be selected using themass spectrometer and dissociated into fragments that can be analyzed byconventional MS, CDMS, or a combination of both conventional MS and CDMSto identify the components present in the selected lipoproteinsubpopulation.

In some embodiments, CDMS may be used in connection with the presentmethods to analyze a broad range of masses encompassing many lipoproteinclasses in a single experiment. For example, CDMS may be employed tocharacterize lipoproteins directly from plasma without the need forextensive isolation or enhancing the purity of the plasma. It should beappreciated that, in some embodiments, CDMS may be used to analyze wholeblood, plasma, or serum to determine abundances of lipoproteinpopulations (e.g., high-density (HDL), low-density (LDL), and verylow-density (VLDL)) and subpopulations. Although whole blood, plasma,and serum are complex mixtures of many different components with widelyvarying concentrations, lipoprotein populations and subpopulations maybe isolated from other components (e.g., proteins) present in the wholeblood, plasma, and serum by correlating the charge, mass, andmass-to-charge (m/z) values measured by CDMS. As discussed furtherbelow, CDMS measurements have shown that the lipoprotein populations andsubpopulations have a range of m/z values.

Definitions

The singular forms “a,” “an” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of the length of a polynucleotide orpolypeptide sequence, dose, time, temperature, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination.

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

As used herein, the terms “reduce,” “reduces,” “reduction” and similarterms mean a decrease of at least about 25%, 35%, 50%, 75%, 80%, 85%,90%, 95%, 97% or more. As used herein, the terms “enhance,” “enhances,”“enhancement” and similar terms indicate an increase of at least about5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

Likewise, an “isolated” lipoprotein means a lipoprotein that is at leastpartially separated from at least some of the other components of thenaturally occurring organism. In representative embodiments an“isolated” lipoprotein is enriched by at least about 10-fold, 100-fold,1000-fold, 10,000-fold or more as compared with the starting material.

As used herein, by “isolate: or “purify” (or grammatical equivalents) alipoprotein, it is meant that the lipoprotein is at least partiallyseparated from at least some of the other components in the startingmaterial. In representative embodiments an “isolated” or “purified”lipoprotein is enriched by at least about 10-fold, 100-fold, 1000-fold,10,000-fold or more as compared with the starting material.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

EXAMPLES

Materials

Low density lipoprotein (LDL), high density lipoprotein (HDL), and HDLsubtypes HDL-2 an HDL-3 were purchased from Academy Bio-Medical Company(Houston, TX, USA). The high density lipoprotein standards were bufferexchanged into 5, 10, or 12.5 mM ammonium acetate (NH₄OAc) at pH 7.5 viamicro Bio-Spin® columns (Bio-Rad Laboratories, Hercules, CA, USA). Lowdensity lipoprotein was dialyzed overnight into 12.5 NH₄OAc at pH 7.5.The final concentration of the lipoprotein standards after exchange intoNH₄OAc was <1 mg/mL.

The lipoprotein standards were purified via ultracentrifugation andisolated from a range of densities. The HDL standard contained allspecies within a density range of 1.063-1.210 g/mL The subtypes of HDL,HDL-2 and HDL-3, were isolated from a smaller band of densities of 1.063to 1.120 g/mL and 1.120 to 1.210 g/mL, respectively.

Instrumentation

The lipoprotein standards were ionized via a commercial nanoelectrosprayionization source before entering a home-built charge detection massspectrometer described in Contino, N. C., et al., Int. J. Mass Spectrom.2013, 345-347, 153-159; Contino, N. C.; et al., J. Am. Soc. MassSpectrom. 2013, 24, 101-108; Pierson, E. E.; et al., Int. J. MassSpectrom. 2013, 337, 50-56; Pierson, E. E.; et al., J. Am. Soc. MassSpectrom. 2015, 26, 1213-1220; Keifer, D. Z.; et al., Anal. Chem. 2015,87, 10330-10337, incorporated by reference herein. Ions entered theinstrument through a heated capillary and were transported through anion funnel, RF hexapole and RF quadrupole. A DC offset on the hexapoleset the nominal ion energy around 100 eV/charge. The ions extracted fromthe quadrupole were focused into the entrance of the dual hemisphericaldeflection energy analyzer (HDA). The energy selected ions were thenfocused into a modified cone trap that contained the charge detectioncylinder. To initiate a trapping event, a potential was placed on therear end cap so that ions were reflected back through the trap. After ashort delay, a potential was placed on the front end cap to close thetrap and caused the ion to oscillate back and forth through the chargedetection cylinder. A trapping period of 100 ms was employed, afterwhich the trap was opened (both end caps set to ground) and the trappingcycle repeated. The ion oscillating back and forth through the detectioncylinder induced a periodic signal which was amplified, digitized, andthen analyzed by a Fortran program using fast Fourier transforms. Them/z was derived from the fundamental frequency and the charge (z) wasderived from the magnitude of the fundamental and first harmonic.

Results

FIGS. 1A-1C show the mass spectra recorded for HDL, subtype HDL-2, andsubtype HDL-3, respectively. Overlaid on each mass spectrum is a scatterplot of m/z (right hand axis) versus mass where each point represents asingle ion. In CDMS, the m/z and mass of individual ions can be directlycorrelated. Clusters of points represent ions that share similar m/zvalues which correspond to particular populations within same subtypethat have slightly different stoichiometry.

The mass spectrum for isolated HDL is shown in FIG. 1A. The mostprominent peak in the mass spectrum was centered at 229 kDa and showed ahigh mass tail that extended up to 1 MDa. Ions were detected out to 3MDa, albeit in low abundance. Without intending to be bound by theory,the CDMS peak shape was expected to be Gaussian with a width determinedby the uncertainties in the m/z and charge measurements. For ahomogenous population of only a single species, the expected peak widthfor the main feature in the mass spectrum would be 17.8 kDa. Instead,the full width at half maximum measured 138 kDa, indicating the peak wasmade up of contributions from many species. The overlaid scatter plot(right hand axis) showed at least four densely populated clusters atwell-defined m/z values of 6.60, 7.50, 8.90, and 10.7 kTh. The centermasses for each subpopulation were determined by isolating the ions ineach cluster, binning the masses into a histogram, and then fitting thehistogram with a Gaussian. Although the ions are well separated by m/z,the four clusters of ions differed only slightly in mass, correspondingto masses of 165, 200, 250 and 376 kDa, respectively.

FIG. 1B illustrates the mass spectrum for subtype HDL-2. The mostabundant feature in the mass spectrum was a peak centered at 238 kDa. Ahigh mass shoulder is present starting around 0.5 MDa and extendedtoward 1 MDa in mass. A low abundant continuum of ions was observed outto 3 MDa. Noise did not contribute to the low abundant ion countsobserved at masses above 1 MDA as empty, partial, and multiple iontrapping events were discarded during data analysis. All ion intensityreported in the mass spectrum were from real ions that had been trappedfor the entire duration of the 100 ms trapping event. The overlaidscatter plot displaying the relationship of m/z versus mass for each ionshowed evidence of at least six distinct subpopulations with m/z below20 kTh. Three densely populated clusters at m/z 6.6, 7.5, and 8.8 kThcontributed to the most abundant feature in the mass spectrum andcorrespond to masses of 161, 197, and 239 kDa, respectively. Twoadditional clusters at 10.1 and 13.4 kTh appeared to contribute to thehigh mass shoulder with the center of masses at 326 and 550 kDa,respectively.

FIG. 1C illustrates the mass spectrum for the smallest of the highdensity lipoprotein subtypes, HDL-3. There were two distinct featuresobserved in the mass spectrum: a high intensity peak centered at 170 kDaand a smaller and a lower abundant feature centered at 340 kDa. The ioncounts diminished as masses reached 2 MDa. Very few ions were presentabove 2 MDa. Eight distinct clusters were observed in the overlaidscatter plot. The four clusters that contained the majority of the totalion intensity were in the low m/z regime positioned at 5.8, 6.8, 7.7,and 9.5 kTh. These clusters correspond to populations with masses of138, 175, 213, and 342 kDa, respectively. The additional high m/zclusters at 11.5, 15.0, and 25.0 kTh correspond to high mass particlescentered around 0.5, 0.9, and 1.1 MDa and were responsible for the broadbut low-abundant distribution of masses that followed the 340 kDa peakin the mass spectrum.

Each subtype, HDL-2 and HDL-3, overlapped in density with the HDLsample, therefore, it is expected that similar subpopulations wereobserved among the three standards. Specifically, HDL and HDL-2 sharedtwo clusters of ions centered around m/z values of 6.5 and 7.5 kThcorresponding to particles having masses of 165 and 200 kDa,respectively. Without intending to be bound by theory, although theexact stoichiometry is unknown, it is likely that these two clusterswere of the same subpopulation in both isolated samples.

The subpopulations with masses of 175, 212, and 342 kDa in FIG. 1C agreewith values estimated from literature and are tentatively assigned tosubpopulations of HDL-3a, HDL-3b, and HDL-3c, respectively. The massesobserved for HDL and HDL-2 subpopulations in FIGS. 1A and 1B do notdirectly conform to particles of known stoichiometry. Currently, morethan 10 HDL subspecies have been identified and are classified bypre-beta and pre-alpha type particles. Without intending to be bound bytheory, it is possible that an assortment of small, medium, and largeHDL particles as well as pre-beta and pre-alpha subspecies were observedby CDMS.

The LDL mass spectrum in FIG. 2 showed a high abundant peak atapproximately 200 kDa followed by a broad distribution of ions centeredaround 2.14 MDa. The overlaid scatter plot showed that the ionscontributing to the 200 kDa peak spanned a range of 5 to 10 kTh with nodiscernible clusters, as were observed in the HDL standards. The ionswith masses centered at 2.14 MDa showed a more disperse distribution ofions with m/z values ranging from 18 to 38 kTh and having a mean valueof 27 kTh. Without intending to be bound by theory, it is likely thehighly disperse range of masses and m/z values for LDL were due todiffering amounts of lipid and cholesterol cargo. There is evidence forsmall, high density LDL where presence is expected to be a biomarker forcoronary artery disease. Ions with masses 0.5 to 2 MDa are tentativelyassigned to the more dense subspecies of LDL.

In addition, a high intensity peak was observed in the LDL standard near200 kDa. Because the structural protein of LDL apolipoprotein B-100weighs in at approximately 550 kDa, the intense low mass peak isattributed to contamination from HDL that may have occurred during theisolation process.

With the direct measurement of mass and known density, the particlediameters of each subtype and subpopulations were estimated. Theparticle diameters were in good agreement with those particle diametersin literature, reporting diameters of 7-12, 9.2-10.2, 7.5-8.8 and 21-27nm for HDL, HDL-2, HDL-3, and LDL, respectively. Although some of thespecies observed in HDL-2 had diameters below 7 nm, those particles canbe attributed to overlap from HDL particles in the isolation process.

FIG. 4A illustrates a CDMS spectrum measured for an HDL sample whereseven subpopulations have been identified in the scatter plot of m/zversus mass. In FIG. 4B, the mass distributions of the subpopulationsidentified in FIG. 4A are plotted along with the overall massdistribution of this HDL sample. The mass distributions for thesubpopulations can be accounted for by HDL particles with differentnumbers of the key structural proteins Apolipoprotein A-1 (Apo A-I) andApolipoprotein A-II (Apo A-II) as described by Lutomski, Gordon,Remaley, and Jarrold (“Resolution of Lipoprotein Subclasses by ChargeDetection Mass Spectrometry”, Anal. Chem. 2018, 90, 6353-6356. DOI:10.1021/acs.analchem.8b01127; the entire contents of which areincorporated by reference herein).

As discussed above, given that HDL is around 50% protein and that thetwo major structural proteins Apo A-I (28,081 Da) and Apo A-II (17,252Da) together contribute around 90% of the total protein, a particle withn_(I) Apo A-I proteins and n_(II) Apo A-II proteins would be expected tohave a mass (in kDa) of aroundm(n _(I) ,n _(II))=(28.081×n _(I)+17.252×n _(II))/0.45  (1)

Masses predicted by Equation 1 are shown by the scales in FIG. 4C. Thelarge digits give the number of Apo A-I proteins, with n_(I) rangingfrom 1 to 6. The smaller digits give the number of Apo A-II proteins,with n_(II) ranging from zero to n_(II)≤n_(I). The points on the scalesgive the masses from Equation 1 for specific combinations of n_(I) andn_(II). For example, for n_(I)=1 and n_(II)=2, the expected mass fromEquation 1 is 163 kDa. A distribution of masses is expected for eachn_(I),n_(II) combination, and the masses given by Equation 1 are onlyapproximate. However, they provide a starting point for assigning thesubpopulations resolved in FIGS. 4A and 4B. For n_(I)=2, there arepoints on the scale in FIG. 4C at 125, 163, and 201 kDa corresponding ton_(II)=0, 1, and 2. These match up with the three lowest mass componentsin FIGS. 4B and 4C (which have average masses of 129/126, 165/163, and200/208 kDa). The resolving power degrades as the mass increases sopeaks due to different n_(II) are not expected to be resolved for highermasses. The resolved component centered on around 250/261 kDa in FIGS.4B and 4C can be attributed to n_(I)=3 and n_(II)=1-2. Similarly, thepeak at 321/327 kDa can be attributed to n_(I)=4 and n_(II)=1-3.Particles with n_(I)=5 and 6 may contribute to the high mass tail on the321-327 kDa peak and to the 511/463 kDa peak. The highest mass componentin FIG. 4 at 756 kDa is large for an HDL particle. A proteomics study ofthe HDL sample revealed the presence of Apo B100, the main structuralprotein of LDL. Thus, an LDL impurity may be responsible for some of thehigh mass component observed in the HDL spectrum.

FIG. 5 illustrates a mass spectrum measured for LDL which shows theresolution of subclasses in the scatter plot of an LDL sample. Incontrast to HDL, where the subclasses have partly discrete massdistributions, the subclasses of LDL have broad and overlapping massdistributions.

What is claimed:
 1. A method of measuring lipoprotein particles of atleast one subtype of a density class of intact lipoprotein particlespresent in a sample containing the density class of intact lipoproteinparticles, the method comprising: a) producing lipoprotein ions from thedensity class of intact lipoprotein particles contained in the sample,b) simultaneously measuring a charge and a mass-to-charge ratio of eachof the lipoprotein ions, c) determining a mass of each of thelipoprotein ions based on the measured charge and mass-to-charge ratiovalues thereof, d) identifying the at least one subtype of the densityclass of intact lipoprotein particles based on the determined masses andmeasured charges of the lipoprotein ions, and e) determining diametersof lipoprotein particles of the identified at least one subtype based onthe determined masses and known densities of the lipoprotein particlesof the identified at least one subtype.
 2. The method of claim 1,wherein the sample contains a plurality of density classes of intactlipoprotein particles, and wherein producing the lipoprotein ionscomprises producing the lipoprotein ions from each of the plurality ofdensity classes of intact lipoprotein particles, and wherein identifyingthe at least one subtype comprises identifying the at least one subtypeof one of the plurality of density classes of intact lipoproteinparticles based on the determined masses and measured charges of thelipoprotein ions.
 3. The method of claim 2, wherein the sample compriseswhole blood, plasma or serum.
 4. The method of claim 2, furthercomprising: (f) identifying at least one subtype of another one of theplurality of density classes of intact lipoprotein particles based onthe determined masses and measured charges of the lipoprotein ions, and(g) determining diameters of lipoprotein particles of the identified atleast one subtype of the another one of the plurality of density classesof intact lipoprotein particles based on the determined masses and knowndensities of the lipoprotein particles of the identified at least onesubtype of the another one of the plurality of density classes of intactlipoprotein particles.
 5. The method of claim 1, wherein the charge andthe mass-to-charge ratio of each of the lipoprotein ions are measuredsimultaneously using charge detection mass spectrometry.
 6. The methodof claim 5, wherein the charge detection mass spectrometry comprises,for each of the lipoprotein ions, focusing the lipoprotein ion into alinear ion trap.
 7. The method of claim 6, wherein the linear ion trapincludes a charge detection cylinder, and wherein the lipoprotein ionoscillates back and forth in the linear ion trap each time passingthrough the charge detection cylinder, and wherein the charge of thelipoprotein ion is measured by measuring a charge induced by thelipoprotein ion on the charge detection cylinder each of multiples timesthe lipoprotein ion passes through the charge detection cylinder, andwherein the mass-to-charge ratio of the lipoprotein ion is measuredbased on a time period spent by the lipoprotein ion each of the multipletimes the lipoprotein ion passes through the charge detection cylinderor a fundamental frequency of oscillation of the lipoprotein ion backand forth through the charge detection cylinder.
 8. A method ofmeasuring lipoprotein particles in at least one subpopulation of asubtype of a density class of intact lipoprotein particles present in asample containing the density class, or the subtype of the densityclass, of intact lipoprotein particles, the method comprising: a)producing lipoprotein ions from the density class, or the subtype of thedensity class, of intact lipoprotein particles contained in the sample,b) simultaneously measuring a charge and a mass-to-charge ratio of eachof the lipoprotein ions, c) determining a mass of each of thelipoprotein ions based on the measured charge and mass-to-charge ratiovalues thereof, d) identifying the at least one subpopulation of thesubtype of the density class of intact lipoprotein particles based onthe determined masses and measured charges of the lipoprotein ions, ande) determining diameters of lipoprotein particles in the identified atleast one subpopulation based on the determined masses and knowndensities of the lipoprotein particles in the identified at least onesubpopulation.
 9. The method of claim 8, wherein the sample contains aplurality of density classes of intact lipoprotein particles, andwherein producing the lipoprotein ions comprises producing thelipoprotein ions from each of the plurality of density classes of intactlipoprotein particles, and wherein identifying the at least onesubpopulation of the subtype of the density class of intact lipoproteinscomprises identifying the at least one subpopulation of the subtype ofone of the plurality of density classes of intact lipoprotein particlesbased on the determined masses and measured charges of the lipoproteinions.
 10. The method of claim 9, wherein the sample comprises wholeblood, plasma or serum.
 11. The method of claim 9, further comprising:(f) identifying at least one subpopulation of the subtype of another oneof the plurality of density classes of intact lipoprotein particlesbased on the determined masses and measured charges of the lipoproteinions, and (g) determining diameters of lipoprotein particles in theidentified at least one subpopulation of the subtype of the another oneof the plurality of density classes of intact lipoprotein particlesbased on the determined masses and known densities of the lipoproteinparticles of the identified at least one subpopulation of the subtype ofthe another one of the plurality of density classes of intactlipoprotein particles.
 12. The method of claim 8, wherein the charge andthe mass-to-charge ratio of each of the lipoprotein ions are measuredsimultaneously using charge detection mass spectrometry.
 13. The methodof claim 12, wherein the charge detection mass spectrometry comprises,for each of the lipoprotein ions, focusing the lipoprotein ion into alinear ion trap.
 14. The method of claim 13, wherein the linear ion trapincludes a charge detection cylinder, and wherein the lipoprotein ionoscillates back and forth in the linear ion trap each time passingthrough the charge detection cylinder, and wherein the charge of thelipoprotein ion is measured based on a charge induced by the lipoproteinion on the charge detection cylinder each of multiple times thelipoprotein ion passes through the charge detection cylinder, andwherein the mass-to-charge ratio of the lipoprotein ion is measuredbased on a time period spent by the lipoprotein ion each of the multipletimes the lipoprotein ion passes through the charge detection cylinderor a fundamental frequency of oscillation of the lipoprotein ion backand forth through the charge detection cylinder.
 15. A method ofidentifying components of lipoprotein particles in a density class,subtype of the density class or subpopulation of the subtype of thedensity class of intact lipoprotein particles present in a samplecontaining the intact lipoprotein particles, the method comprising: a)producing lipoprotein ions from the intact lipoprotein particlescontained in the sample, b) measuring mass-to-charge ratios of thelipoprotein ions, c) selecting a subset of the measured lipoprotein ionshaving mass-to-charge ratios within a selected range of mass-to-chargeratios, d) dissociating the lipoprotein ions in the selected subset intofragment ions, and e) simultaneously measuring a charge and amass-to-charge ratio of the fragment ions; f) determining masses of thefragment ions based on the measured charge and mass-to-charge ratiovalues thereof; and g) identifying the components of the lipoproteinparticles in the density class, subtype of the density class orsubpopulation of the subtype of the density class of intact lipoproteinparticles based on the determined masses of the fragment ions.
 16. Themethod of claim 15, wherein selecting the subset of the measuredlipoprotein ions comprises selecting, based on the measuredmass-to-charge ratios of the lipoprotein ions, a subset of the measuredlipoprotein ions that corresponds to the subtype of the density class ofthe intact lipoprotein particles.
 17. The method of claim 15, whereinselecting the subset of the measured lipoprotein ions comprisesselecting, based on the measured mass-to-charge ratios of thelipoprotein ions, a subset of the measured lipoprotein ions thatcorresponds to the subpopulation of the subtype of the density class ofthe intact lipoprotein particles.
 18. The method of claim 15, whereinthe sample comprises whole blood, plasma or serum.
 19. The method ofclaim 15, wherein the charge and the mass-to-charge ratio of each of thefragment ions are measured simultaneously using charge detection massspectrometry, and wherein the charge detection mass spectrometrycomprises, for each of the fragment ions, focusing the fragment ion intoa linear ion trap.
 20. The method of claim 19, wherein the linear iontrap includes a charge detection cylinder, and wherein the fragment ionoscillates back and forth in the linear ion trap each time passingthrough the charge detection cylinder, and wherein the charge of thefragment ion is measured based on a charge induced by the fragment ionon the charge detection cylinder each of multiple times the fragment ionpasses through the charge detection cylinder, and wherein themass-to-charge ratio of the fragment ion is measured based on a timeperiod spent by the fragment ion each of the multiple times the fragmention passes through the charge detection cylinder or a fundamentalfrequency of oscillation of the fragment ion back and forth through thecharge detection cylinder.